The proposed study has grown from a collaboration of the Sharpless & Taylor laboratories to employ novel "click chemistry" to develop selective ligands for nicotinic acetylcholine receptor using the acetylcholine binding protein as the reaction vessel. The precursors are anchored to two sites on the receptor and contain extended azide and acetylene moieties that react by cycloaddition to form a triazole. The precursor ligands have modest affinity for the protein and react extremely slowly in solution. On the protein surface, the apposition of the reactant groups and the amphiphilic environment of the protein forces a rapid reaction leading to formation of high affinity complex. Synthesis employs combinatorial reactants, and a single or predominant product is formed that is characterized by DIOS mass spectrometry. Interestingly, this "freezeframe" procedure catches the complex in unanticipated conformations such that the high affinity regioisomer forms from a low abundance conformation. Initially acetylcholine binding proteins from Lymnaea and Aplysia are employed as templates to form selective nicotinic agents. By mutagenesis the template is modified to incorporate binding determinants of the a7 receptor and then the heterologous receptor subtypes found in the brain. Since this is effectively a "freeze-frame" reaction, conformational flexibility of the binding templates will be analyzed by decay of fluorescence anisotropy in separate studies. Complexes also offer the potential for X-ray crystallographic studies. Partal agonists and antagonists will be generated from combinatorial libraries using nicotine, epibatidine or other heterocycles or bicyclic rings as the active center and peripheral site anchoring loci. Cycloaddition products will be examined for affinity and specificity for the respective binding templates. Their selectivities will then be examined on nicotinic receptors formed from transfected receptor subunit combinations. The final stages of investigation will include studies in rodents to determine efficacy and toxicity.